Laboratories in many biomedical specialties, such as anatomic pathology, hematology, and microbiology, examine tissue under a microscope for the presence and the nature of disease. In recent years, these laboratories have shown a growing interest in microscopic digital imaging as an adjunct to direct visual examination. Digital imaging has a number of advantages including the ability to document disease, share findings, collaborate (as in telemedicine), and analyze morphologic findings by computer. Though numerous studies have shown that digital image quality is acceptable for most clinical and research use, some aspects of microscopic digital imaging are limited in application.
Perhaps the most important limitation to microscopic digital imaging is a “sub-sampling” problem encountered in all single frame images. The sub-sampling problem has two components: a field of view problem and a resolution-based problem. The field of view problem limits an investigator looking at a single frame because what lies outside the view of an image on a slide cannot be determined. The resolution-based problem occurs when the investigator looking at an image is limited to viewing a single resolution of the image. The investigator cannot “zoom in” for a closer examination or “zoom out” for a bird's eye view when only a single resolution is available. Significantly, the field of view and resolution-based problems are inversely related. Thus, as one increases magnification to improve resolution, one decreases the field of view. For example, as a general rule, increasing magnification by a factor of two decreases the field of view by a factor of four.
To get around the limitations of single frame imaging, developers have looked at two general options. The first option takes the form of “dynamic-robotic” imaging, in which a video camera on the microscope transmits close to real time images to the investigator looking at a monitor, while the investigator operates the microscope by remote control. These systems have been used successfully in initial telepathology collaborations by allowing a distant consultant to view the specimen without the delays and losses associated with sending the physical slide to the consultant for review, and by allowing the consultant to view the entire slide, not just a few static images captured by the initial user.
However, these systems do not lend themselves to significant collaborations, documentation or computer based analysis. To be successful, remote transmission requires lossy video compression techniques to be used in order to meet the network bandwidth requirements, or requires significant delays in the image display if lossless transmission is used. In addition, lossy compression on the order required for real-time remote transmission, severely limits computer-based analysis, as well as human diagnosis, due to the artifacts associated with lossy compression techniques. Remote operation of a microscope also requires only a single user to use the instrument at one time, requiring instrument scheduling and local maintenance of the instrument and the slides to be viewed.
The second option being investigated to overcome the limitations inherit in single frame imaging is a montage (or “virtual slide”) approach. In this method, a robotic microscope systematically scans the entire slide, taking an image at each “camera field” corresponding to the field of view of the camera. Camera field and field of view shall hereinafter be referred to as the “field.” The individual images are then “knitted” together in a software application to form a very large data set with very appealing properties. The robotic microscope can span the entire slide area at a resolution limited only by the power of the optical system and camera. Software exists to display this data set at any resolution on a computer screen, allowing the user to zoom in, zoom out, and pan around the data set as if using a physical microscope. The data set can be stored for documentation, shared over the Internet, or analyzed by computer programs.
The “virtual slide” option has some limitations, however. One of the limitations is file size. For an average tissue section, the data generated at 0.33 μm/pixel can be between two and five gigabytes uncompressed. In an extreme case, the data generated from one slide can be up to thirty-six gigabytes.
A much more difficult limitation with the prior systems is an image capture time problem. Given an optical primary magnification of twenty and a two-third inch coupled device or “CCD”, the system field of view is approximately (8.8 mm×6.6 mm)/20=0.44×0.33 mm. A standard tissue section of approximately 2.25 square centimeters, therefore, requires approximately fifteen hundred fields to capture an image of the entire tissue section.
Field rate, which is the amount of time it takes to capture an image of a field and set-up the apparatus capturing the field for a following image capture, in montage systems is limited by three factors—camera frame rate (the number of camera images acquired per second), image processing speed (including any time required to read the camera data, perform any processing on the camera data prior to storage, and to store the final data), and rate of slide motion, which is the time required for the slide to be mechanically repositioned for the next image acquisition. Given today's technology, the rate of slide motion is a significant limiting factor largely because the existing imaging systems require the slide to come to a stop at the center of each field to capture a blur free image of the field.
For example, traditional bright field microscopic illumination systems were designed to support direct visual examination of a specimen on the field and therefore depend on a continuous light source for illumination. Continuous light, however, is a significant limitation for digital imaging in that the slide, which must move to capture an entire image, but must be stationary with respect to the camera during CCD integration, thus moving the slide from the light. Moreover, slide motion during integration results in a blurred image. Traditional montage systems, therefore, have had to move the slide (and stage) from field to field in a precise “move, stop, take image and move again” pattern. This pattern requires precise, expensive mechanics, and its speed is inherently limited by the inertia of the stage.
The three-dimensional characteristic of a typical tissue sample and the slide places additional limitations on the imaging system. Like all lenses, microscope optics have a finite depth of field—the distance within which objects will appear to be focused. A typical depth of field is about 8 microns for a 10× objective, and in general, as the magnification increases, the depth of field decreases. While microscope slides are polished glass, the flatness of the slide can vary on the order of 50 microns or more across the slide. The variations in the tissue sample thickness and any defects associated with placing the sample on the slide, such as folds in the tissue, also affect the optimal position of the slide with respect to the imaging optics. The magnitude of the optimal position and the limited depth of field of the microscope optics require the focus to be adjusted as the system moves from field to field. The time to refocus the system at each field also contributes to the overall capture time of the montage image.
Thus, a system is needed to reduce the image capture time. The system must also enable efficient and high quality imaging of a microscope slide via a high-resolution slide scanning process.